Human amine receptor

ABSTRACT

A Human amine receptor polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also provided are methods for detecting compounds which bind to and activate and bind to and inhibit such polypeptide and the use of compounds for treating diseases related to the under-expression and over-expression of the Human amine receptor of the present invention. Also disclosed are methods for detecting mutations in the nucleic acid sequence encoding the polypeptide and for detecting altered levels of the soluble form of the polypeptide.

[0001] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as well as the production of suchpolynucleotides and polypeptides. More particularly, the polypeptide ofthe present invention are human 7-transmembrane receptors and has beenputatively identified as a human amine receptor. The invention alsorelates to inhibiting the action of such polypeptides.

[0002] It is well established that many medically significant biologicalprocesses are mediated by proteins participating in signal transductionpathways that involve G-proteins and/or second messengers, e.g., cAMP(Lefkowitz, Nature, 351:353-354 (1991)). Herein these proteins arereferred to as proteins participating in pathways with G-proteins or PPGproteins. Some examples of these proteins include the GPC receptors suchas those for adrenergic agents and dopamine (Kobilka, B. K., et al.,PNAS, 84:46-50 (1987); Kobilka, B. K., et al., Science, 238:650-656(1987); Bunzow, J. R., et al., Nature, 336:783-787 (1988)), G-proteinsthemselves, effector proteins, e.g., phospholipase C, adenyl cyclase,and phosphodiesterase, and actuator proteins, e.g., protein kinase A andprotein kinase C (Simon, M. I., et al., Science, 252:802-8 (1991)).

[0003] For example, in one form of signal transduction, the effect ofhormone binding is activation of an enzyme, adenylate cyclase, insidethe cell. Enzyme activation by hormones is dependent on the presence ofthe nucleotide GTP, and GTP also influences hormone binding. A G-proteinconnects the hormone receptors to adenylate cyclase. G-protein was shownto exchange GTP for bound GDP when activated by hormone receptors. TheGTP-carrying form then binds to an activated adenylate cyclase.Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns theG-protein to its basal, inactive form. Thus, the G-protein serves a dualrole, as an intermediate that relays the signal from receptor toeffector, and as a clock that controls the duration of the signal.

[0004] The membrane protein gene superfamily of G-protein coupledreceptors has been characterized as having seven putative transmembranedomains. The domains are believed to represent transmembrane a-helicesconnected by extracellular or cytoplasmic loops. G-protein coupledreceptors include a wide range of biologically active receptors, such ashormone, viral, growth factor and neuroreceptors.

[0005] G-protein coupled receptors can be intracellularly coupled byheterotrimeric G-proteins to various intracellular enzymes, ion channelsand transporters (see, Johnson et al., Endoc., Rev., 10:317-331 (1989)).Different G-protein α-subunits preferentially stimulate particulareffectors to modulate various biological functions in a cell.Phosphorylation of cytoplasmic residues of G-protein coupled receptorshave been identified as an important mechanism for the regulation ofG-protein coupling of some G-protein coupled receptors. G-proteincoupled receptors are found in numerous sites within a mammalian host.

[0006] The Human Amine Receptor of the present invention is a G-proteincoupled receptor. Neurosensory and neuromotor functions are carried outby neurotransmission. Neurotransmission is the conductance of a nerveimpulse from one neuron, called the presynaptic neuron, to anotherneuron, called the postsynaptic neuron, across the synaptic cleft.Transmission of the nerve impulse across the synaptic cleft involves thesecretion of neurotransmitter substances. The neurotransmitter ispackaged into vesicles in the presynaptic neuron and released into thesynaptic cleft to find its receptor at the postsynaptic neuron.Transmission of the nerve impulse is normally transient.

[0007] An essential property of synaptic transmission is the rapidtermination of action following neurotransmitter release. For manyneurotransmitters, including catecholamine, serotonin, and certain aminoacids (e.g., gamma-aminobutyric acid (GABA), glutamate and glycine),rapid termination of synaptic action is achieved by the uptake of theneurotransmitter into the presynaptic terminal and surrounding glialcells. This rapid re-accumulation of a neurotransmitter is the result ofre-uptake by the presynaptic terminals.

[0008] At presynaptic terminals, the various molecular structures forre-uptake are highly specific for such neurotransmitters as choline andthe biogenic amines (low molecular weight neurotransmitter substancessuch as dopamine, norepinephrine, epinephrine, serotonin and histamine).These molecular apparatuses are receptors which are termed transporters.These transporters move neurotransmitter substances from the synapticcleft back across the cell membrane of the presynaptic neuron into thecytoplasm of the presynaptic terminus and therefore terminate thefunction of these substances. Inhibition or stimulation ofneurotransmitter uptake provides a means for modulating the effects ofthe endogenous neurotransmitters.

[0009] The neurotransmitter substances are implicated in numerouspathiophysiologies and treatments including, movement disorders,schizophrenia, drug addiction, anxiety, migraine headaches, epilepsy,myocloinus, spastic paralysis, muscle spasm, schizophrenia, cognitiveimpairment, depression, Parkinson's Disease and Alzheimer's Disease,among others.

[0010] Re-uptake of neurotransmitter substances by the transporters maybe sodium-dependent. For instance, the GABA transporter is a member ofthe recently described sodium-dependent neurotransmitter transportergene family. These transporters are transmembrane receptor complexeshaving an extracellular portion, a transmembrane portion and anintracellular portion. A significant degree of homology exists in thetransmembrane domains of the entire family of sodium-dependentneurotransmitter transporter proteins, with considerable stretches ofidentical amino acids, while much less homology is apparent in theintracellular and extracellular loops connecting these domains. Theextracellular loop in particular seems to be unique for eachtransporter. This region may contribute to substrate and/or inhibitorspecificities.

[0011] The polypeptide of the present invention has been putativelyidentified as an amine receptor. This identification has been made as aresult of amino acid sequence homology to the rat amine receptor.

[0012] In accordance with one aspect of the present invention, there areprovided novel mature receptor polypeptides as well as biologicallyactive and diagnostically or therapeutically useful fragments, analogsand derivatives thereof. The receptor polypeptides of the presentinvention are of human origin.

[0013] In accordance with another aspect of the present invention, thereare provided isolated nucleic acid molecules encoding the receptorpolypeptides of the present invention, including mRNAs, DNAs, cDNAs,genomic DNA as well as antisense analogs thereof and biologically activeand diagnostically or therapeutically useful fragments thereof.

[0014] In accordance with a further aspect of the present invention,there are provided processes for producing such receptor polypeptides byrecombinant techniques comprising culturing recombinant prokaryoticand/or eukaryotic host cells, containing nucleic acid sequences encodingthe receptor polypeptides of the present invention, under conditionspromoting expression of said polypeptides and subsequent recovery ofsaid polypeptides.

[0015] In accordance with yet a further aspect of the present invention,there are provided antibodies against such receptor polypeptides.

[0016] In accordance with another aspect of the present invention thereare provided methods of screening for compounds which bind to andactivate or inhibit activation of the receptor polypeptides of thepresent invention.

[0017] In accordance with still another embodiment of the presentinvention there are provided processes of administering compounds to ahost which bind to and activate the receptor polypeptide of the presentinvention for the prevention and/or treatment of abnormal conditionsresulting from under-expression of the amino receptor of the presentinvention.

[0018] In accordance with another aspect of the present invention thereis provided a method of administering the receptor polypeptides of thepresent invention via gene therapy to treat conditions related tounder-expression of the polypeptide or underexpression of a ligand tothe receptor polypeptide.

[0019] In accordance with still another embodiment of the presentinvention there are provided processes of administering compounds whichbind to and inhibit activation of the receptor polypeptides of thepresent invention for prevention and/or treatment of conditionsresulting from expression of the amine receptor of the presentinvention.

[0020] In accordance with yet another aspect of the present invention,there are provided nucleic acid probes comprising nucleic acid moleculesof sufficient length to specifically hybridize to the polynucleotidesequences of the present invention.

[0021] In accordance with still another aspect of the present invention,there are provided diagnostic assays for detecting diseases related tomutations in the nucleic acid sequences encoding such polypeptides andfor detecting an altered level of the soluble form of the receptorpolypeptides.

[0022] In accordance with yet a further aspect of the present invention,there are provided processes for utilizing such receptor polypeptides,or polynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

[0023] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

[0024] The following drawings are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

[0025]FIG. 1 illustrates the cDNA sequence and corresponding deducedamino acid sequence of the human amine receptor of the presentinvention. The standard one-letter abbreviations for amino acids areused. Sequencing was performed using a 373 Automated DNA sequencer(Applied Biosystems, Inc.).

[0026]FIG. 2 is an illustration of an amino acid homology alignmentbetween the amine transporter or the present invention (top line) andmurine β-1 Adrenoreceptor (bottom line).

[0027]FIG. 3 is an illustration of an amino acid homology alignmentbetween the amine transporter or the present invention (top line) andhuman dopamine D2 receptor (bottom line).

[0028] The amine receptor of the present invention may be responsiblefor re-uptake of one or any of the amine neurotransmitters present inmammalian cells. Examples of such amine transporters include, but arenot limited to, dopamine, norepinephrine, epinephrine, serotonin andhistamine, and other amino acid transmitters, including GABA, glycineand glutamate.

[0029] In accordance with an aspect of the present invention, there isprovided an isolated nucleic acid (polynucleotide) which encodes for themature polypeptide having the deduced amino acid sequence of FIG. 1 (SEQID NO:2) or for the mature polypeptide encoded by the cDNA of the clonedeposited as ATCC Deposit No. ______ on Jun. 1, 1995.

[0030] A polynucleotide encoding a polypeptide of the present inventionmay be found in human monocytes. The polynucleotide of this inventionwas discovered in a human genomic library. It is structurally related tothe G protein-coupled receptor family. It contains an open reading frameencoding a protein of 337 amino acid residues. The protein exhibits thehighest degree of homology to a murine β-1 Adrenoreceptor with 32.099%identity and 55.864% similarity over a 330 amino acid stretch. Theprotein also exhibits homology to a human dopamine D₂ receptor with 32%identity and 58.333% similarity over a 312 amino acid stretch.

[0031] The polynucleotide of the present invention may be in the form ofRNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptide may beidentical to the coding sequence shown in FIG. 1 (SEQ ID NO:1) or thatof the deposited clone or may be a different coding sequence whichcoding sequence, as a result of the redundancy or degeneracy of thegenetic code, encodes the same mature polypeptide as the DNA of FIG. 1(SEQ ID NO:1) or the deposited CDNA.

[0032] The polynucleotide which encodes for the mature polypeptide ofFIG. 1 (SEQ ID NO:2) or for the mature polypeptide encoded by thedeposited cDNA may include: only the coding sequence for the maturepolypeptide; the coding sequence for the mature polypeptide andadditional coding sequence; the coding sequence for the maturepolypeptide (and optionally additional coding sequence) and non-codingsequence, such as introns or non-coding sequence 5′ and/or 3′ of thecoding sequence for the mature polypeptide.

[0033] Thus, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes only coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

[0034] The present invention further relates to variants of thehereinabove described polynucleotides which encode for fragments,analogs and derivatives of the polypeptide having the deduced amino acidsequence of FIG. 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNAof the deposited clone. The variant of the polynucleotide may be anaturally occurring allelic variant of the polynucleotide or anon-naturally occurring variant of the polynucleotide.

[0035] Thus, the present invention includes polynucleotides encoding thesame mature polypeptide as shown in FIG. 1 (SEQ ID NO:2) or the samemature polypeptide encoded by the cDNA of the deposited clone as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptide of FIG. 1 (SEQ ID NO:2) or thepolypeptide encoded by the cDNA of the deposited clone. Such nucleotidevariants include deletion variants, substitution variants and additionor insertion variants.

[0036] As hereinabove indicated, the polynucleotide may have a codingsequence which is a naturally occurring allelic variant of the codingsequence shown in FIG. 1 (SEQ ID NO:1) or of the coding sequence of thedeposited clone. As known in the art, an allelic variant is an alternateform of a polynucleotide sequence which may have a substitution,deletion or addition of one or more nucleotides, which does notsubstantially alter the function of the encoded polypeptide.

[0037] The polynucleotides may also encode for a soluble form of theamine receptor polypeptide which is the extracellular portion of thepolypeptide which has been cleaved from the TM and intracellular domainof the full-length polypeptide of the present invention.

[0038] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

[0039] Fragments of the full length gene of the present invention may beused as a hybridization probe for a cDNA library to isolate the fulllength cDNA and to isolate other cDNAs which have a high sequencesimilarity to the gene or similar biological activity. Probes of thistype preferably have at least 30 bases and may contain, for example, 50or more bases. The probe may also be used to identify a cDNA clonecorresponding to a full length transcript and a genomic clone or clonesthat contain the complete gene including regulatory and promotorregions, exons, and introns. An example of a screen comprises isolatingthe coding region of the gene by using the known DNA sequence tosynthesize an oligonucleotide probe. Labeled oligonucleotides having asequence complementary to that of the gene of the present invention areused to screen a library of human cDNA, genomic DNA or mRNA to determinewhich members of the library the probe hybridizes to.

[0040] The present invention further relates to polynucleotides whichhybridize to the hereinabove-described sequences if there is at least70%, preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIG. 1 (SEQ ID NO:1) orthe deposited cDNA(s), i.e. function as a soluble amine receptor byretaining the ability to bind the ligands for the receptor even thoughthe polypeptide does not function as a membrane bound amine receptor,for example, by eliciting a second messenger response.

[0041] Alternatively, the polynucleotides may have at least 20 bases,preferably 30 bases and more preferably at least 50 bases whichhybridize to a polynucleotide of the present invention and which have anidentity thereto, as hereinabove described, and which may or may notretain activity. Such polynucleotides may be employed as probes for thepolynucleotide of SEQ ID NO: 1, or for variants thereof, for example,for recovery of the polynucleotide or as a diagnostic probe or as a PCRprimer.

[0042] Thus, the present invention is directed to polynucleotides havingat least a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 as well as fragments thereof, which fragments have atleast 30 bases and preferably at least 50 bases and to polypeptidesencoded by such polynucleotides.

[0043] The deposit(s) referred to herein will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for purposes of Patent Procedure. Thesedeposits are provided merely as convenience to those of skill in the artand are not an admission that a deposit is required under 35 U.S.C.§112. The sequence of the polynucleotides contained in the depositedmaterials, as well as the amino acid sequence of the polypeptidesencoded thereby, are incorporated herein by reference and arecontrolling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

[0044] The present invention further relates to a human amine receptorpolypeptide which has the deduced amino acid sequence of FIG. 1 (SEQ IDNo. 2) or which has the amino acid sequence encoded by the depositedcDNA, as well as fragments, analogs and derivatives of such polypeptide.

[0045] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptide of FIG. 1 (SEQ ID No. 2) or that encoded by thedeposited cDNA, means a polypeptide which retains essentially the samebiological function or activity as such polypeptide, i.e. functions asan amine receptor, or retains the ability to bind the ligand for thereceptor even though the polypeptide does not function as a G-proteincoupled receptor, for example, a soluble form of the receptor.

[0046] The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

[0047] The fragment, derivative or analog of the polypeptide of FIG. 1(SEQ ID No. 2) or that encoded by the deposited CDNA may be (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which b one ormore of the amino acid residues includes a substituent group, or (iii)one in which the mature polypeptide is fused with another compound, suchas a compound to increase the half-life of the polypeptide (for example,polyethylene glycol) or (iv) one in which the additional amino acids arefused to the mature polypeptide which are employed for purification ofthe mature polypeptide or (v) one in which a fragment of the polypeptideis soluble, i.e. not membrane bound, yet still binds ligands to themembrane bound receptor. Such fragments, derivatives and analogs aredeemed to be within the scope of those skilled in the art from theteachings herein.

[0048] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0049] The polypeptides of the present invention include the polypeptideof SEQ ID NO:2 (in particular the mature polypeptide) as well aspolypeptides which have at least 70% similarity (preferably at least 70%identity) to the polypeptide of SEQ ID NO:2 and more preferably at least90% similarity (more preferably at least 90% identity) to thepolypeptide of SEQ ID NO:2 and still more preferably at least 95%similarity (still more preferably at least 95% identity) to thepolypeptide of SEQ ID NO:2 and to portions of such polypeptide with suchportion of the polypeptide generally contains at least 30 amino acidsand more preferably at least 50 amino acids.

[0050] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and conserved amino acidsubstitutes thereto of the polypeptide to the sequence of a secondpolypeptide.

[0051] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis, therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention.

[0052] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region “leader and trailer” as well as intervening sequences(introns) between individual coding segments (exons).

[0053] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

[0054] The polypeptides of the present invention include the polypeptideof SEQ ID NO:2 (in particular the mature polypeptide) as well aspolypeptides which have at least 70% similarity (preferably at least 70%identity) to the polypeptide of SEQ ID NO:2 and more preferably at least90% similarity (more preferably at least 90% identity) to thepolypeptide of SEQ ID NO:2 and still more preferably at least 95%similarity (still more preferably at least 95% identity) to thepolypeptide of SEQ ID NO:2 and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids.

[0055] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0056] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention.

[0057] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0058] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes of the present invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

[0059] The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

[0060] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0061] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or trip, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0062] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0063] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

[0064] As representative examples of appropriate hosts, there may bementioned: bacterial cells, such as E. coli, Streptomyces, Salmonellatyphimurium; fungal cells, such as yeast; insect cells such asDrosophila S2 and Spodoptera Sf9; animal cells such as CHO, HEK, COS orBowes melanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

[0065] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, PRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, PBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

[0066] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are PKK232-8 and PCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0067] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

[0068] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0069] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), the disclosure of which is hereby incorporated byreference.

[0070] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0071] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences. Optionally, the heterologous sequence can encodea fusion protein including an N-terminal identification peptideimparting desired characteristics, e.g., stabilization or simplifiedpurification of expressed recombinant product.

[0072] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0073] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0074] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0075] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0076] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well know to those skilled in the art.

[0077] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HEK, HeLa and BHKcell lines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

[0078] The human amine receptor polypeptide can be recovered andpurified from recombinant cell cultures by methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Protein refolding steps can beused, as necessary, in completing configuration of the mature protein.Finally, high performance liquid chromatography (HPLC) can be employedfor final purification steps.

[0079] The polypeptides of the present invention may be a naturallypurified product, or a product of chemical synthetic procedures, orproduced by recombinant techniques from a prokaryotic or eukaryotic host(for example, by bacterial, yeast, higher plant, insect and mammaliancells in culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

[0080] Fragments of the full length human amine transporter gene may beused as a hybridization probe for a cDNA library to isolate the fulllength gene and to isolate other genes which have a high sequencesimilarity to the gene or similar biological activity. Probes of thistype are at least 20 bases, preferably at least 30 bases and mostpreferably at least 50 bases or more. The probe may also be used toidentify a cDNA clone corresponding to a full length transcript and agenomic clone or clones that contain the complete human aminetransporter gene including regulatory and promotor regions, exons, andintrons. As an example of a screen comprises isolating the coding regionof the human amine transporter gene by using the known DNA sequence tosynthesize an oligonucleotide probe. Labeled oligonucleotides having asequence complementary to that of the gene of the present invention areused to screen a library of human CDNA, genomic DNA or mRNA to determinewhich members of the library the probe hybridizes to.

[0081] This invention provides a method for determining amineneurotransmitters which are transported by the human amine receptor ofthe present invention. An example of an assay which will identify theseneurotransmitters comprises infecting mammalian cells with recombinantvaccinia virus strain VTF-7 encoding a T7 RNA polymerase and followingsuch infection with liposome-mediated transfection with the aminereceptor gene of the present invention through the use of a vector, forexample, pBSSKII(−). Controlled transfections are also done withequivalent amounts of vector alone. Assays are performed eight hoursfollowing transfection in modified Krebs-Ringer-HEPES buffer. Cells arethen incubated with [³H] neurotransmitter (for example, GABA, dopamine,serotonin, etc.). Uptake is stopped by placing the cells on ice. Cellsare solubilized in one percent SDS, and the amount of radioactivityaccumulated is determined by liquid scintillation counting. Asignificant amount of uptake determines that the particularneurotransmitter is taken up by the human amine receptor of the presentinvention by determining background using control transfections withPBSSKII for each assay and subtracting the values obtained from thesignals determined for the specific amine neurotransmitters.

[0082] This invention also provides a method of detecting expression ofthe amine receptor of the present invention on the surface of a cell bydetecting the presence of mRNA coding for the amine receptor. Thismethod comprises obtaining total mRNA from the cell using methodswell-known in the art and contacting the mRNA so obtained with a nucleicacid probe of at least 10 nucleotides and which is capable ofspecifically hybridizing with a sequence included within the sequence ofa nucleic acid molecule encoding a human amine receptor of the presentinvention under hybridizing conditions, detecting the presence of mRNAhybridized to the probe, and thereby detecting the expression of theamine receptor by the cell. Hybridization of probes to target nucleicacid molecules such as mRNA molecules employs techniques well known inthe art. However, in one embodiment of this invention, nucleic acids areextracted by precipitation from lysed cells and the mRNA is isolatedfrom the extract using a column which binds the poly-A tails of the mRNAmolecules. The mRNA is then exposed to radioactively labelled probe on anitrocellulose membrane, and the probe hybridizes to and thereby labelscomplementary mRNA sequences. Binding may be detected by autoradiographyor scintillation counting. However, other methods for performing thesesteps are well known to those of skill in the art.

[0083] Alternatively, an antibody directed to the human amine receptormay be employed under conditions permitting binding of the antibody tothe transporter, and detecting the presence of the receptor on thesurface of the cell. Such a method may be employed for determiningwhether a given cell is defective in expression of the amine receptor.Detection methods include fluorescent markers bound to the antibodies.

[0084] The invention also provides a method for determining whether acompound not known to be capable of specifically binding to a humanamine receptor can specifically bind to the human amine receptor, whichcomprises contacting a mammalian cell comprising a plasmid adapted forexpression in a mammalian cell which plasmid further comprises a DNAwhich expresses the amine receptor on the cell surface with the compoundunder conditions permitting binding of ligands known to bind to theamine receptor, detecting the presence of any compound bound to theamine receptor, the presence of bound compound indicating that thecompound is capable of specifically binding to the human amine receptor.

[0085] The polynucleotides and polypeptides of the present invention maybe employed as research reagents and materials for discovery oftreatments and diagnostics to human disease.

[0086] The amine receptor of the present invention may be employed in aprocess for screening for compounds which activate (agonists) or inhibitactivation (antagonists) of the receptor polypeptide of the presentinvention In general, such screening procedures involve providingappropriate cells which express the receptor polypeptide of the presentinvention on the surface thereof. Such cells include cells from mammals,yeast, drosophila or E. Coli. In particular, a polynucleotide encodingthe receptor of the present invention is employed to transfect cells tothereby express the amine receptor. The expressed receptor is thencontacted with a test compound to observe binding, stimulation orinhibition of a functional response.

[0087] One such screening procedure involves the use of melanophoreswhich are transfected to express the amine receptor of the presentinvention. Such a screening technique is described in PCT WO 92/01810published Feb. 6, 1992.

[0088] Thus, for example, such assay may be employed for screening for acompound which inhibits activation of the receptor polypeptide of thepresent invention by contacting the melanophore cells which encode thereceptor with both the receptor ligand and a compound to be screened.Inhibition of the signal generated by the ligand indicates that acompound is a potential antagonist for the receptor, i.e., inhibitsactivation of the receptor.

[0089] The screen may be employed for determining a compound whichactivates the receptor by contacting such cells with compounds to bescreened and determining whether such compound generates a signal, i.e.,activates the receptor.

[0090] Other screening techniques include the use of cells which expressthe amine receptor (for example, transfected CHO cells) in a systemwhich measures extracellular pH changes caused by receptor activation,for example, as described in Science, volume 246, pages 181-296 (October1989). For example, compounds may be contacted with a cell whichexpresses the receptor polypeptide of the present invention and a secondmessenger response, e.g. signal transduction or pH changes, may bemeasured to determine whether the potential compound activates orinhibits the receptor.

[0091] Another such screening technique involves introducing RNAencoding the amine receptor into Xenopus oocytes to transiently expressthe receptor. The receptor oocytes may then be contacted with thereceptor ligand and a compound to be screened, followed by detection ofinhibition or activation of a calcium signal in the case of screeningfor compounds which are thought to inhibit activation of the receptor.

[0092] Another screening technique involves expressing the aminereceptor in which the receptor is linked to a phospholipase C or D. Asrepresentative examples of such cells, there may be mentionedendothelial cells, smooth muscle cells, embryonic kidney cells, etc. Thescreening may be accomplished as hereinabove described by detectingactivation of the receptor or inhibition of activation of the receptorfrom the phospholipase second signal.

[0093] Another method involves screening for compounds which inhibitactivation of the receptor polypeptide of the present inventionantagonists by determining inhibition of binding of labeled ligand tocells which have the receptor on the surface thereof. Such a methodinvolves transfecting a eukaryotic cell with DNA encoding the aminereceptor such that the cell expresses the receptor on its surface andcontacting the cell with a compound in the presence of a labeled form ofa known ligand. The ligand can be labeled, e.g., by radioactivity. Theamount of labeled ligand bound to the receptors is measured, e.g., bymeasuring radioactivity of the receptors. If the compound binds to thereceptor as determined by a reduction of labeled ligand which binds tothe receptors, the binding of labeled ligand to the receptor isinhibited.

[0094] Amine receptors are ubiquitous in the mammalian host and areresponsible for many biological functions, including many pathologies.Accordingly, it is desirous to find compounds and drugs which stimulatethe amine receptor on the one hand and which can inhibit the function ofa amine receptor on the other hand.

[0095] Examples of compounds which bind to and inhibit the aminereceptor of the present invention includes antibodies, or in some casesan oligopeptides, which bind to the amine receptor but do not elicit asecond messenger response such that the activity of the amine receptoris prevented. Antibodies include anti-idiotypic antibodies whichrecognize unique determinants generally associated with theantigen-binding site of an antibody.

[0096] Another example includes proteins which are closely related tothe ligand of the amine receptors, i.e. a fragment of the ligand, whichhas lost biological function and when binding to the amine receptor,elicits no response.

[0097] An antisense construct prepared through the use of antisensetechnology, may be used to control gene expression through triple-helixformation or antisense DNA or RNA, both of which methods are based onbinding of a polynucleotide to DNA or RNA. For example, the 5′ codingportion of the polynucleotide sequence, which encodes for the maturepolypeptides of the present invention, is used to design an antisenseRNA oligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix-see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervanet al., Science, 251:1360 (1991)), thereby preventing transcription andthe production of the amine receptor. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of mRNA moleculesinto amine receptor (antisense—Okano, J. Neurochem., 56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). The oligonucleotides described abovecan also be delivered to cells such that the antisense RNA or DNA may beexpressed in vivo to inhibit production of the amine receptor.

[0098] A small molecule which binds to the amine receptor, making itinaccessible to ligands such that normal biological activity isprevented, for example small peptides or peptide-like molecules, mayalso be used to inhibit activation of the receptor polypeptide of thepresent invention.

[0099] A soluble form of the amine receptor, e.g. a fragment of thereceptor, may be used to inhibit activation of the receptor by bindingto the ligand to the receptor polypeptide of the present invention andpreventing the ligand from interacting with membrane bound aminereceptors.

[0100] This invention additionally provides a method of treating anabnormal condition related to expression of the amine receptor of thepresent invention which comprises administering to a subject aninhibitory compound as hereinabove described along with apharmaceutically acceptable carrier in an amount effective to block bindto a human amine receptor can specifically bind to the human aminereceptor, which comprises contacting a mammalian cell comprising aplasmid adapted for expression in a mammalian cell which plasmid furthercomprises a DNA which expresses the amine receptor on the cell surfacewith the compound under conditions permitting binding of ligands knownto bind to the amine receptor, detecting the presence of any compoundbound to the amine receptor, the presence of bound compound indicatingthat the compound is capable of specifically binding to the human aminereceptor.

[0101] The polynucleotides and polypeptides of the present invention maybe employed as research reagents and materials for discovery oftreatments and diagnostics to human disease.

[0102] The amine receptor of the present invention may be employed in aprocess for screening for compounds which activate (agonists) or inhibitactivation (antagonists) of the receptor polypeptide of the presentinvention

[0103] In general, such screening procedures involve providingappropriate cells which express the receptor polypeptide of the presentinvention on the surface thereof. Such cells include cells from mammals,yeast, drosophila or E. Coli. In particular, a polynucleotide encodingthe receptor of the present invention is employed to transfect cells tothereby express the amine receptor. The invention also provides apharmaceutical pack or kit comprising one or more containers filled withone or more of the ingredients of the pharmaceutical compositions of theinvention. Associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. In addition, the pharmaceutical compositions may beemployed in conjunction with other therapeutic compounds.

[0104] The pharmaceutical compositions may be administered in aconvenient manner such as by the oral, topical, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal or intradermalroutes. The pharmaceutical compositions are administered in an amountwhich is effective for treating and/or prophylaxis of the specificindication. In general, they are administered in an amount of at leastabout 10 μg/kg body weight and in most cases they will be administeredin an amount not in excess of about 8 mg/Kg body weight per day. In mostcases, the dosage is from about 10 μg/kg to about 1 mg/kg body weightdaily, taking into account the routes of administration, symptoms, etc.

[0105] The human amine receptor and agonist and antagonist compoundswhich are polypeptides may also be employed in accordance with thepresent invention by expression of such polypeptides in vivo, which isoften referred to as “gene therapy.”

[0106] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

[0107] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

[0108] Retroviruses from which the retroviral plasmid vectorshereinabove mentioned may be derived include, but are not limited to,Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0109] The vector includes one or more promoters. Suitable promoterswhich may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990(1989), or any other promoter (e.g., cellular promoters such aseukaryotic cellular promoters including, but not limited to, thehistone, pol III, and β-actin promoters). Other viral promoters whichmay be employed include, but are not limited to, adenovirus promoters,thymidine kinase (TK) promoters, and B19 parvovirus promoters. Theselection of a suitable promoter will be apparent to those skilled inthe art from the teachings contained herein.

[0110] The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe genes encoding the polypeptides.

[0111] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein byreference in its entirety. The vector may transduce the packaging cellsthrough any means known in the art. Such means include, but are notlimited to, electroporation, the use of liposomes, and CaPO₄precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

[0112] The producer cell line generates infectious retroviral vectorparticles which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

[0113] This invention is also related to the use of the human aminereceptor gene as part of a diagnostic assay for detecting diseases orsusceptibility to diseases related to the presence of mutations in thehuman amine receptor genes. Such diseases are related tounder-expression of the human amine receptor.

[0114] Individuals carrying mutations in the human amine receptor genemay be detected at the DNA level by a variety of techniques. Nucleicacids for diagnosis may be obtained from a patient's cells, such as fromblood, urine, saliva, tissue biopsy and autopsy material. The genomicDNA may be used directly for detection or may be amplified enzymaticallyby using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior toanalysis. RNA or CDNA may also be used for the same purpose. As anexample, PCR primers complementary to the nucleic acid encoding thehuman amine receptor protein can be used to identify and analyze humanamine receptor mutations. For example, deletions and insertions can bedetected by a change in size of the amplified product in comparison tothe normal genotype. Point mutations can be identified by hybridizingamplified DNA to radiolabeled human amine receptor RNA or alternatively,radiolabeled human amine receptor antisense DNA sequences. Perfectlymatched sequences can be distinguished from mismatched duplexes by RNaseA digestion or by differences in melting temperatures.

[0115] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

[0116] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401(1985)).

[0117] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

[0118] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0119] The present invention also relates to a diagnostic assay fordetecting altered levels of soluble forms of the amine receptorpolypeptides of the present invention in various tissues which may beemployed to diagnose diseases related to under-expression of the aminereceptor. Assays used to detect levels of the soluble receptorpolypeptides in a sample derived from a host are well known to those ofskill in the art and include radioimmunoassays, competitive-bindingassays, Western blot analysis and preferably as ELISA assay.

[0120] An ELISA assay initially comprises preparing an antibody specificto antigens of the amine receptor polypeptides, preferably a monoclonalantibody. In addition a reporter antibody is prepared against themonoclonal antibody. To the reporter antibody is attached a detectablereagent such as radioactivity, fluorescence or in this example ahorseradish peroxidase enzyme. A sample is now removed from a host andincubated on a solid support, e.g. a polystyrene dish, that binds theproteins in the sample. Any free protein binding sites on the dish arethen covered by incubating with a non-specific protein such as bovineserum albumin. Next, the monoclonal antibody is incubated in the dishduring which time the monoclonal antibodies attach to any amine receptorproteins attached to the polystyrene dish. All unbound monoclonalantibody is washed out with buffer. The reporter antibody linked tohorseradish peroxidase is now placed in the dish resulting in binding ofthe reporter antibody to any monoclonal antibody bound to amine receptorproteins. Unattached reporter antibody is then washed out. Peroxidasesubstrates are then added to the dish and the amount of color developedin a given time period is a measurement of the amount of amine receptorproteins present in a given volume of patient sample when comparedagainst a standard curve.

[0121] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

[0122] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the CDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

[0123] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0124] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For a review of this technique, see Verma etal., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press,New York (1988).

[0125] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0126] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0127] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0128] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0129] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0130] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,1975, Nature, 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies(Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96).

[0131] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice may be used to express humanized antibodies toimmunogenic polypeptide products of this invention.

[0132] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0133] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0134] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0135] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0136] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res., 8:4057 (1980).

[0137] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0138] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., Id., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units of T4 DNAligase (“ligase”) per 0.5 μg of approximately equimolar amounts of theDNA fragments to be ligated.

[0139] Unless otherwise stated, transformation was performed asdescribed in the method of Graham, F. and Van der Eb, A., Virology,52:456-457 (1973).

EXAMPLE 1 Bacterial Expression and Purification of Human Amine Receptor

[0140] The DNA sequence encoding human amine receptor, ATCC #______, isinitially amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ end sequences of the processed amine receptor nucleic acidsequence (minus the signal peptide sequence). Additional nucleotidescorresponding to amine receptor gene are added to the 5′ and 3′sequences respectively. The 5′ oligonucleotide primer has the sequence5′ CGGAATTCCTUATGAGAGCTGTCTTCATC 3′ (SEQ ID No. 3) contains an EcoRIrestriction enzyme site followed by 18 nucleotides of human aminereceptor coding sequence starting from the presumed terminal amino acidof the processed protein. The 3′ sequence 5′CGGAAGCTTCGTCATTCTTGGTACAAATCAAC 3′ (SEQ ID No. 4) containscomplementary sequences to an HindIII site and is followed by 18nucleotides of the human amine receptor gene. The restriction enzymesites correspond to the restriction enzyme sites on the bacterialexpression vector pQE-9 (Qiagen, Inc. Chatsworth, Calif.). pQE-9 encodesantibiotic resistance (Amp^(r)), a bacterial origin of replication(ori), an IPTG-regulatable promoter operator (P/O), a ribosome bindingsite (RBS), a 6-His tag and restriction enzyme sites. pQE-9 is thendigested with HindIII and EcoRI. The amplified sequences are ligatedinto pQE-9 and are inserted in frame with the sequence encoding for thehistidine tag and the RBS. The ligation mixture is then used totransform E. coli strain M15/rep 4 (Qiagen, Inc.) by the proceduredescribed in Sambrook, J. et al., Molecular Cloning: A LaboratoryManual, Cold Spring Laboratory Press, (1989). M15/rep4 contains multiplecopies of the plasmid pREP4, which expresses the lacI repressor and alsoconfers kanamycin resistance (Kan^(r)). Transformants are identified bytheir ability to grow on LB plates and ampicillin/kanamycin resistantcolonies are selected. Plasmid DNA is isolated and confirmed byrestriction analysis. Clones containing the desired constructs are grownovernight (O/N) in liquid culture in LB media supplemented with both Amp(100 μg/ml) and Kan (25 μg/ml). The O/N culture is used to inoculate alarge culture at a ratio of 1:100 to 1:250. The cells are grown to anoptical density 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(“Isopropyl-B-D-thiogalacto pyranoside”) is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression. Cells are grownan extra 3 to 4 hours. Cells are then harvested by centrifugation. Thecell pellet is solubilized in the chaotropic agent 6 Molar GuanidineHCl. After clarification, solubilized human amine receptor is purifiedfrom this solution by chromatography on a Nickel-Chelate column underconditions that allow for tight binding by proteins containing the 6-Histag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984)). Humanamine receptor protein is eluted from the column in 6 molar guanidineHCl pH 5.0 and for the purpose of renaturation adjusted to 3 molarguanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione (reduced)and 2 mmolar glutathione (oxidized). After incubation in this solutionfor 12 hours the protein is dialyzed to 10 mmolar sodium phosphate.

EXAMPLE 2 Cloning and Expression of Human Amine Receptor Using theBaculovirus Expression System

[0141] The DNA sequence encoding the full length human amine receptorprotein, ATCC #____, is amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene:

[0142] The 5′ primer has the sequence 5′ 5′ CGGGATCCCTCCATGAGAGCTGTCTTCATC 3′ (SEQ ID No. 5) and contains a BamHI restriction enzymesite followed by 4 nucleotides resembling an efficient signal for theinitiation of translation in eukaryotic cells (Kozak, M., J. Mol. Biol.,196:947-950 (1987) which is just behind the first 18 nucleotides of thehuman amine receptor gene.

[0143] The 3′ primer has the sequence 5′ CGGGATCCCGCTCATTCTTGG TACAAATC3′ (SEQ ID No. 6) and contains the cleavage site for the restrictionendonuclease BamHI and 18 nucleotides complementary to the 3′non-translated sequence of the human amine receptor gene. The amplifiedsequences are isolated from a 1% agarose gel using a commerciallyavailable kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). Thefragment is then digested with the endonucleases BamHI and then purifiedagain on a 1% agarose gel. This fragment is designated F2.

[0144] The vector pRG1 (modification of pVL941 vector, discussed below)is used for the expression of the human amine receptor protein using thebaculovirus expression system (for review see: Summers, M. D. and Smith,G. E. 1987, A manual of methods for baculovirus vectors and insect cellculture procedures, Texas Agricultural Experimental Station Bulletin No.1555). This expression vector contains the strong polyhedrin promoter ofthe Autographa californica nuclear polyhedrosis virus (AcMNPV) followedby the recognition sites for the restriction endonucleases BamHI. Thepolyadenylation site of the simian virus (SV)40 is used for efficientpolyadenylation. For an easy selection of recombinant viruses thebeta-galactosidase gene from E. coli is inserted in the same orientationas the polyhedrin promoter followed by the polyadenylation signal of thepolyhedrin gene. The polyhedrin sequences are flanked at both sides byviral sequences for the cell-mediated homologous recombination ofco-transfected wild-type viral DNA. Many other baculovirus vectors couldbe used in place of pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summers, M. D., Virology, 170:31-39).

[0145] The plasmid is digested with the restriction enzymes BamHI andthen dephosphorylated using calf intestinal phosphatase by proceduresknown in the art. The DNA is then isolated from a 1% agarose gel usingthe commercially available kit (“Geneclean” BIO 101 Inc., La Jolla,Calif.). This vector DNA is designated V2.

[0146] Fragment F2 and the dephosphorylated plasmid V2 are ligated withT4 DNA ligase. E. coli HB101 cells are then transformed and bacteriaidentified that contained the plasmid (pBac-Human amine receptor) withthe human amine receptor gene using the enzyme BamHI. The sequence ofthe cloned fragment is confirmed by DNA sequencing.

[0147] 5 μg of the plasmid pBac-Human amine receptor is co-transfectedwith 1.0 μg of a commercially available linearized baculovirus(“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.) using thelipofection method (Felgner et al. Proc. Natl. Acad. Sci. USA,84:7413-7417 (1987)).

[0148] 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac-Humanamine receptor are mixed in a sterile well of a microtiter platecontaining 50 μl of serum free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to the Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27° C. After 5 hours the transfection solution is removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. The plate is put back into an incubator and cultivationcontinued at 27° C. for four days.

[0149] After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

[0150] Four days after the serial dilution, the viruses are added to thecells and blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses is then resuspendedin an Eppendorf tube containing 200 μl of Grace's medium. The agar isremoved by a brief centrifugation and the supernatant containing therecombinant baculovirus is used to infect Sf9 cells seeded in 35 mmdishes. Four days later the supernatants of these culture dishes areharvested and then stored at 4° C.

[0151] Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-Human amine receptor at a multiplicity of infection (MOI)of 2. Six hours later the medium is removed and replaced with SF900 IImedium minus methionine and cysteine (Life Technologies Inc.,Gaithersburg). 42 hours later 5 μCi of ³⁵S-methionine and 5 μCi ³⁵Scysteine (Amersham) are added. The cells are further incubated for 16hours before they are harvested by centrifugation and the labelledproteins visualized by SDS-PAGE and autoradiography.

EXAMPLE 3 Expression of Recombinant Human Amine Receptor in COS Cells

[0152] The expression of plasmid, Human amine receptor HA is derivedfrom a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin ofreplication, 2) ampicillin resistance gene, 3) E. coli replicationorigin, 4) CMV promoter followed by a polylinker region, a SV40 intronand polyadenylation site. A DNA fragment encoding the entire Human aminereceptor precursor and a HA tag fused in frame to its 3′ end is clonedinto the polylinker region of the vector, therefore, the recombinantprotein expression is directed under the CMV promoter. The HA tagcorrespond to an epitope derived from the influenza hemagglutininprotein as previously described (I. Wilson, et al., Cell, 37:767,(1984)). The infusion of HA tag to the target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

[0153] The plasmid construction strategy is described as follows:

[0154] The DNA sequence encoding Human amine receptor, ATCC #______, isconstructed by PCR using two primers: the 5′ primer 5′GTCCAAGCTTGCCACCATGAGAGCTGTCTTCATC 3′ (SEQ ID No. 7) contains a HindIIIsite followed by 18 nucleotides of Human amine receptor coding sequencestarting from the initiation codon; the 3′ sequence 5′CTAGCTCGAGTCAAGCGTA GTCTGGGACGTCGTATGGGTAGCATTCTTGGTACAAATCAAC 3′ (SEQID No. 8) contains complementary sequences to an XhoI site, translationstop codon, HA tag and the last 18 nucleotides of the Human aminereceptor coding sequence (not including the stop codon). Therefore, thePCR product contains a HindIII site, human amine receptor codingsequence followed by HA tag fused in frame, a translation terminationstop codon next to the HA tag, and an HindIII site. The PCR amplifiedDNA fragment and the vector, pcDNAI/Amp, are digested with HindIII andXhoI restriction enzymes and ligated. The ligation mixture istransformed into E. coli strain SURE (Stratagene Cloning Systems, LaJolla, Calif.) the transformed culture is plated on ampicillin mediaplates and resistant colonies are selected. Plasmid DNA is isolated fromtransformants and examined by restriction analysis for the presence ofthe correct fragment. For expression of the recombinant amine receptor,COS cells are transfected with the expression vector by DEAE-DEXTRANmethod (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: ALaboratory Manual, Cold Spring Laboratory Press, (1989)). The expressionof the Human amine receptor HA protein is detected by radiolabelling andimmunoprecipitation method (E. Harlow, D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelledfor 8 hours with ³⁵S-cysteine two days post transfection. Culture mediais then collected and cells are lysed with detergent (RIPA buffer (150mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5 DOC, 50 mM Tris, pH 7.5)(Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culturemedia are precipitated with a HA specific monoclonal antibody. Proteinsprecipitated are analyzed on 15% SDS-PAGE gels.

EXAMPLE 4 Expression Pattern of Human Amine Receptor in Human Tissue

[0155] Northern blot analysis is carried out to examine the levels ofexpression of Human amine receptor in human tissues. Total cellular RNAsamples are isolated with RNAzol™ B system (Biotecx Laboratories, Inc.Houston, Tex.). About 1μg of total RNA isolated from each human tissuespecified is separated on 1% agarose gel and blotted onto a nylon filter(Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring HarborPress, (1989)). The labeling reaction is done according to theStratagene Prime-It kit with 50 ng DNA fragment. The labeled DNA ispurified with a Select-G-50 column (5 Prime-3 Prime, Inc. Boulder,Colo.). The filter is then hybridized with radioactive labeled fulllength Human amine receptor gene at 1,000,000 cpm/ml in 0.5 M NaPO₄, pH7.4 and 7% SDS overnight at 65° C. After wash twice at room temperatureand twice at 6° C. with 0.5× SSC, 0.1% SDS, the filter is then exposedat −70° C. overnight with an intensifying screen.

EXAMPLE 5 Expression via Gene Therapy

[0156] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

[0157] pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked bythe long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0158] The cDNA encoding a polypeptide of the present invention isamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively. The 5′ primer contains an EcoRI site and the 3′primer contains a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the EcoRI and HindIII fragment areadded together, in the presence of T4 DNA ligase. The resulting mixtureis maintained under conditions appropriate for ligation of the twofragments. The ligation mixture is used to transform bacteria HB101,which are then plated onto agar-containing kanamycin for the purpose ofconfirming that the vector had the gene of interest properly inserted.

[0159] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells are transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[0160] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his.

[0161] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product.

[0162] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1 10 1380 base pairs nucleic acid double linear DNA (genomic) CDS252..1262 1 CTAGAGCTAG CAGGAGTAAC TCTCATGGAA CCTTGGAAAC CATTCTTCAATTGAATTTCA 60 GGGCACATTT GAATCAGTAC CCAGGGGCAC TGTACTATGC TCCCAGCTGGACCTTAGTTT 120 CCTCCTCCTC GTTTCACCCT GTGAGTAATT AACAGACAAA ATTTTTTTTTTTTTTTTTTT 180 TTTTTTTTTT TTTTTGCCCT CCAGTGGAGA AGGTGGCCAG TTCTCAGACAGAGGAAGAGT 240 AGAAATCATA A ATG AGA GCT GTC TTC ATC CAA GGT GCT GAA GAGCAC CCT 290 Met Arg Ala Val Phe Ile Gln Gly Ala Glu Glu His Pro 1 5 10GCG GCA TTC TGC TAC CAG GTG AAT GGG TCT TGC CCC AGG ACA GTA CAT 338 AlaAla Phe Cys Tyr Gln Val Asn Gly Ser Cys Pro Arg Thr Val His 15 20 25 ACTCTG GGC ATC CAG TTG GTC ATC TAC CTG ACC TGT GCA GCA GGC ATG 386 Thr LeuGly Ile Gln Leu Val Ile Tyr Leu Thr Cys Ala Ala Gly Met 30 35 40 45 CTGATT ATC GTG CTA GGG AAT GTA TTT GTG GCA TTT GCT GTG TCC TAC 434 Leu IleIle Val Leu Gly Asn Val Phe Val Ala Phe Ala Val Ser Tyr 50 55 60 TTC AAAGCG CTT CAC ACG CCC ACC AAC TTC CTG CTG CTC TCC CTG GCC 482 Phe Lys AlaLeu His Thr Pro Thr Asn Phe Leu Leu Leu Ser Leu Ala 65 70 75 CTG GCT GACATG TTT CTG GGT CTG CTG GTG CTG CCC CTC AGC ACC ATT 530 Leu Ala Asp MetPhe Leu Gly Leu Leu Val Leu Pro Leu Ser Thr Ile 80 85 90 CGC TCA GTG GAGAGC TGC TGG TTC TTC GGG GAC TTC CTC TGC CGC CTG 578 Arg Ser Val Glu SerCys Trp Phe Phe Gly Asp Phe Leu Cys Arg Leu 95 100 105 CAC ACC TAC CTGGAC ACC CTC TTC TGC CTC ACC TCC ATC TTC CAT CTC 626 His Thr Tyr Leu AspThr Leu Phe Cys Leu Thr Ser Ile Phe His Leu 110 115 120 125 TGT TTC ATTTCC ATT GAC CGC CAC TGT GCC ATC TGT GAC CCC CTG CTC 674 Cys Phe Ile SerIle Asp Arg His Cys Ala Ile Cys Asp Pro Leu Leu 130 135 140 TAT CCC TCCAAG TTC ACA GTG AGG GTG GCT CTC AGG TAC ATC CTG GCA 722 Tyr Pro Ser LysPhe Thr Val Arg Val Ala Leu Arg Tyr Ile Leu Ala 145 150 155 GGA TGG GGGGTG CCC GCA GCA TAC ACT TCG TTA TTC CTC TAC ACA GAT 770 Gly Trp Gly ValPro Ala Ala Tyr Thr Ser Leu Phe Leu Tyr Thr Asp 160 165 170 GTG GTA GAGACA AGG CTC AGC CAG TGG CTG GAA GAG ATG CCT TGT GTG 818 Val Val Glu ThrArg Leu Ser Gln Trp Leu Glu Glu Met Pro Cys Val 175 180 185 GGC AGT TGCCAG CTG CTG CTC AAT AAA TTT TGG GGC TGG TTA AAC TTC 866 Gly Ser Cys GlnLeu Leu Leu Asn Lys Phe Trp Gly Trp Leu Asn Phe 190 195 200 205 CCT TTGTTC TTT GTC CCC TGC CTC ATT ATG ATC AGC TTG TAT GTG AAG 914 Pro Leu PhePhe Val Pro Cys Leu Ile Met Ile Ser Leu Tyr Val Lys 210 215 220 ATC TTTGTG GTT GCT ACC AGA CAG GCT CAG CAG ATT ACC ACA TTG AGC 962 Ile Phe ValVal Ala Thr Arg Gln Ala Gln Gln Ile Thr Thr Leu Ser 225 230 235 AAA AGCCTG GCT GGG GCT GCC AAG CAT GAG AGA AAA GCT GCC AAG ACC 1010 Lys Ser LeuAla Gly Ala Ala Lys His Glu Arg Lys Ala Ala Lys Thr 240 245 250 CTG GGCATT GTT GTG GGC ATA TAC CTC TTG TGC TGG CTG CCC TTC ACC 1058 Leu Gly IleVal Val Gly Ile Tyr Leu Leu Cys Trp Leu Pro Phe Thr 255 260 265 ATA GACACG ATG GTC GAC AGC CTC CTT CAC TTT ATC ACA CCC CCA CTG 1106 Ile Asp ThrMet Val Asp Ser Leu Leu His Phe Ile Thr Pro Pro Leu 270 275 280 285 GTCTTT GAC ATC TTT ATC TGG TTT GCT TAC TTC AAC TCA GCC TGC AAC 1154 Val PheAsp Ile Phe Ile Trp Phe Ala Tyr Phe Asn Ser Ala Cys Asn 290 295 300 CCCATC ATC TAT GTC TTT TCC TAC CAG TGG TTT CGG AAG GCA CTG AAA 1202 Pro IleIle Tyr Val Phe Ser Tyr Gln Trp Phe Arg Lys Ala Leu Lys 305 310 315 CTCACA CTG AGC CAG AAG GTC TTC TCA CCG CAG ACA CGC ACT GTT GAT 1250 Leu ThrLeu Ser Gln Lys Val Phe Ser Pro Gln Thr Arg Thr Val Asp 320 325 330 TTGTAC CAA GAA TGATTCCTTC TACTAAATGC AGGCAAGGAG TAGGACCTCA 1302 Leu Tyr GlnGlu 335 CAGGAAAGAT AAGTGGCACT GTGACCGCGG GCTGTGTGGT GTTGAGTTTGTGGGCATGCT 1362 TCCAGGACAG CATGGGTT 1380 337 amino acids amino acidlinear protein 2 Met Arg Ala Val Phe Ile Gln Gly Ala Glu Glu His Pro AlaAla Phe 1 5 10 15 Cys Tyr Gln Val Asn Gly Ser Cys Pro Arg Thr Val HisThr Leu Gly 20 25 30 Ile Gln Leu Val Ile Tyr Leu Thr Cys Ala Ala Gly MetLeu Ile Ile 35 40 45 Val Leu Gly Asn Val Phe Val Ala Phe Ala Val Ser TyrPhe Lys Ala 50 55 60 Leu His Thr Pro Thr Asn Phe Leu Leu Leu Ser Leu AlaLeu Ala Asp 65 70 75 80 Met Phe Leu Gly Leu Leu Val Leu Pro Leu Ser ThrIle Arg Ser Val 85 90 95 Glu Ser Cys Trp Phe Phe Gly Asp Phe Leu Cys ArgLeu His Thr Tyr 100 105 110 Leu Asp Thr Leu Phe Cys Leu Thr Ser Ile PheHis Leu Cys Phe Ile 115 120 125 Ser Ile Asp Arg His Cys Ala Ile Cys AspPro Leu Leu Tyr Pro Ser 130 135 140 Lys Phe Thr Val Arg Val Ala Leu ArgTyr Ile Leu Ala Gly Trp Gly 145 150 155 160 Val Pro Ala Ala Tyr Thr SerLeu Phe Leu Tyr Thr Asp Val Val Glu 165 170 175 Thr Arg Leu Ser Gln TrpLeu Glu Glu Met Pro Cys Val Gly Ser Cys 180 185 190 Gln Leu Leu Leu AsnLys Phe Trp Gly Trp Leu Asn Phe Pro Leu Phe 195 200 205 Phe Val Pro CysLeu Ile Met Ile Ser Leu Tyr Val Lys Ile Phe Val 210 215 220 Val Ala ThrArg Gln Ala Gln Gln Ile Thr Thr Leu Ser Lys Ser Leu 225 230 235 240 AlaGly Ala Ala Lys His Glu Arg Lys Ala Ala Lys Thr Leu Gly Ile 245 250 255Val Val Gly Ile Tyr Leu Leu Cys Trp Leu Pro Phe Thr Ile Asp Thr 260 265270 Met Val Asp Ser Leu Leu His Phe Ile Thr Pro Pro Leu Val Phe Asp 275280 285 Ile Phe Ile Trp Phe Ala Tyr Phe Asn Ser Ala Cys Asn Pro Ile Ile290 295 300 Tyr Val Phe Ser Tyr Gln Trp Phe Arg Lys Ala Leu Lys Leu ThrLeu 305 310 315 320 Ser Gln Lys Val Phe Ser Pro Gln Thr Arg Thr Val AspLeu Tyr Gln 325 330 335 Glu 29 base pairs nucleic acid single linear DNA(genomic) 3 CGGAATTCCT UATGAGAGCT GTCTTCATC 29 32 base pairs nucleicacid single linear DNA (genomic) 4 CGGAAGCTTC GTCATTCTTG GTACAAATCA AC32 30 base pairs nucleic acid single linear DNA (genomic) 5 CGGGATCCCTCCATGAGAGC TGTCTTCATC 30 29 base pairs nucleic acid single linear DNA(genomic) 6 CGGGATCCCG CTCATTCTTG GTACAAATC 29 34 base pairs nucleicacid single linear DNA (genomic) 7 GTCCAAGCTT GCCACCATGA GAGCTGTCTT CATC34 61 base pairs nucleic acid single linear DNA (genomic) 8 CTAGCTCGAGTCAAGCGTAG TCTGGGACGT CGTATGGGTA GCATTCTTGG TACAAATCAA 60 C 61 365 aminoacids amino acid Not Relevant Not Relevant protein 9 Ala Arg Leu Leu ValLeu Ala Ser Pro Pro Ala Ser Leu Leu Pro Pro 1 5 10 15 Ala Ser Glu GlySer Ala Pro Leu Ser Gln Gln Trp Thr Ala Gly Met 20 25 30 Gly Leu Leu ValAla Leu Ile Val Leu Leu Ile Val Val Gly Asn Val 35 40 45 Leu Val Ile ValAla Ile Ala Lys Thr Pro Arg Leu Gln Thr Leu Thr 50 55 60 Asn Leu Phe IleMet Ser Leu Ala Ser Ala Asp Leu Val Met Gly Leu 65 70 75 80 Leu Val ValPro Phe Gly Ala Thr Ile Val Val Trp Gly Arg Trp Glu 85 90 95 Tyr Gly SerPhe Phe Cys Glu Leu Trp Thr Ser Val Asp Val Leu Cys 100 105 110 Val ThrAla Ser Ile Glu Thr Leu Cys Val Ile Ala Leu Asp Arg Tyr 115 120 125 LeuAla Ile Thr Ser Pro Phe Arg Tyr Gln Ser Leu Leu Thr Arg Ala 130 135 140Arg Ala Arg Ala Leu Val Cys Thr Val Trp Ala Ile Ser Ala Leu Val 145 150155 160 Ser Phe Leu Pro Ile Leu Met His Trp Trp Arg Ala Glu Ser Asp Glu165 170 175 Ala Arg Arg Cys Tyr Asn Asp Pro Lys Cys Cys Asp Phe Val ThrAsn 180 185 190 Arg Ala Tyr Ala Ile Ala Ser Ser Val Val Ser Phe Tyr ValPro Leu 195 200 205 Cys Ile Met Ala Phe Val Tyr Leu Arg Val Phe Arg GluAla Gln Lys 210 215 220 Gln Val Lys Lys Ile Asp Ser Cys Glu Arg Arg PheLeu Gly Gly Pro 225 230 235 240 Ala Arg Pro Pro Ser Pro Glu Pro Ser ProSer Pro Gly Pro Pro Arg 245 250 255 Pro Ala Asp Ser Leu Ala Asn Gly ArgSer Ser Lys Arg Arg Pro Ser 260 265 270 Arg Leu Val Ala Leu Arg Glu GlnLys Ala Leu Lys Thr Leu Gly Ile 275 280 285 Ile Met Gly Val Phe Thr LeuCys Trp Leu Pro Phe Phe Leu Ala Asn 290 295 300 Val Val Lys Ala Phe HisArg Asp Leu Val Pro Asp Arg Leu Phe Val 305 310 315 320 Phe Phe Asn TrpLeu Gly Tyr Ala Asn Ser Ala Phe Asn Pro Ile Ile 325 330 335 Tyr Cys ArgSer Pro Asp Phe Arg Lys Ala Phe Gln Arg Leu Leu Cys 340 345 350 Cys AlaArg Arg Ala Ala Cys Arg Arg Arg Ala Ala His 355 360 365 353 amino acidsamino acid Not Relevant linear protein 10 Asp Asp Asp Leu Glu Arg GlnAsn Trp Ser Arg Pro Phe Asn Gly Ser 1 5 10 15 Asp Gly Lys Ala Asp ArgPro His Tyr Asn Tyr Tyr Ala Thr Leu Leu 20 25 30 Thr Leu Leu Ile Ala ValIle Val Phe Gly Asn Val Leu Val Cys Met 35 40 45 Ala Val Ser Arg Glu LysAla Leu Gln Thr Thr Thr Asn Tyr Leu Ile 50 55 60 Val Ser Leu Ala Val AlaAsp Leu Leu Val Ala Thr Leu Val Met Pro 65 70 75 80 Trp Val Val Tyr LeuGlu Val Val Gly Glu Trp Lys Phe Ser Arg Ile 85 90 95 His Cys Asp Ile PheVal Thr Leu Asp Val Met Met Cys Thr Ala Ser 100 105 110 Ile Leu Asn LeuCys Ala Ile Ser Ile Asp Arg Tyr Thr Ala Val Ala 115 120 125 Met Pro MetLeu Tyr Asn Thr Arg Tyr Ser Ser Lys Arg Arg Val Thr 130 135 140 Val MetIle Ser Ile Val Trp Val Leu Ser Phe Thr Ile Ser Cys Pro 145 150 155 160Leu Leu Phe Gly Leu Asn Asn Ala Asp Gln Asn Glu Cys Ile Ile Ala 165 170175 Asn Pro Ala Phe Val Val Tyr Ser Ser Ile Val Ser Phe Tyr Val Pro 180185 190 Phe Ile Val Thr Leu Leu Val Tyr Ile Lys Ile Tyr Ile Val Leu Arg195 200 205 Arg Arg Arg Lys Arg Val Asn Thr Lys Arg Ser Ser Arg Ala PheArg 210 215 220 Ala His Leu Arg Ala Pro Leu Lys Glu Ala Ala Arg Arg GluLys Asn 225 230 235 240 Gly His Ala Lys Asp His Pro Lys Ile Ala Lys IlePhe Glu Ile Gln 245 250 255 Thr Met Pro Asn Gly Lys Thr Arg Thr Ser LeuLys Thr Met Ser Arg 260 265 270 Arg Lys Leu Ser Gln Gln Lys Glu Lys LysAla Thr Gln Met Leu Ala 275 280 285 Ile Val Leu Gly Val Phe Ile Ile CysTrp Leu Pro Phe Phe Ile Thr 290 295 300 His Ile Leu Asn Ile His Cys AspCys Asn Ile Pro Pro Val Leu Tyr 305 310 315 320 Ser Ala Phe Thr Trp LeuGly Tyr Val Asn Ser Ala Val Asn Pro Ile 325 330 335 Ile Tyr Thr Thr PheAsn Ile Glu Phe Arg Lys Ala Phe Leu Lys Ile 340 345 350 Leu

What is claimed is:
 1. An isolated polynucleotide comprising a memberselected from the group consisting of: (a) a polynucleotide encoding thepolypeptide as set forth in FIG.
 1. (b) a polynucleotide encoding thepolypeptide expressed by the DNA contained in ATCC Deposit No. ______;(c) a polynucleotide capable of hybridizing to and which is at least 70%identical to the polynucleotide of (a) or (b); and (d) a polynucleotidefragment of the polynucleotide of (a), (b) or (c).
 2. The polynucleotideof claim 1 encoding the polypeptide of FIG.
 1. 3. The polynucleotide ofclaim 1 wherein said polynucleotide encodes a mature polypeptide encodedby the DNA contained in ATCC Deposit No. ______.
 4. A vector containingthe polynucleotide of claim
 1. 5. A host cell genetically engineeredwith the vector of claim
 4. 6. A process for producing a polypeptidecomprising: expressing from the host cell of claim 5 the polypeptideencoded by said polynucleotide.
 7. A process for producing cells capableof expressing a polypeptide comprising genetically engineering cellswith the vector of claim
 4. 8. A polypeptide selected from the groupconsisting of (i) a polypeptide having the deduced amino acid sequenceof FIG. 1 and fragments, analogs and derivatives thereof; and (ii) apolypeptide encoded by the CDNA of ATCC Deposit No. ______ andfragments, analogs and derivatives of said polypeptide.
 9. Thepolypeptide of claim 8 wherein the polypeptide has the deduced aminoacid sequence of FIG.
 1. 10. An antibody against the polypeptide ofclaim
 8. 11. A compound which activates the polypeptide of claim
 8. 12.A compound which inhibits activation of the polypeptide of claim
 8. 13.A method for the treatment of a patient having need to activate areceptor comprising: administering to the patient a therapeuticallyeffective amount of the compound of claim
 11. 14. A method for thetreatment of a patient having need to inhibit a receptor comprising:administering to the patient a therapeutically effective amount of thecompound of claim
 12. 15. The method of claim 13 wherein said compoundis a polypeptide and a therapeutically effective amount of the compoundis administered by providing to the patient DNA encoding said agonistand expressing said agonist in vivo.
 16. The method of claim 14 whereinsaid compound is a polypeptide and a therapeutically effective amount ofthe compound is administered by providing to the patient DNA encodingsaid antagonist and expressing said antagonist in vivo.
 17. A method foridentifying a compound which bind to and activate the polypeptide ofclaim 8 comprising: contacting a compound with cells expressing on thesurface thereof the polypeptide of claim 8, said polypeptide beingassociated with a second component capable of providing a detectablesignal in response to the binding of a compound to said polypeptide saidcontacting being under conditions sufficient to permit binding ofcompounds to the polypeptide; and identifying a compound capable ofpolypeptide binding by detecting the signal produced by said secondcomponent.
 18. A method for identifying compounds which bind to andinhibit activation of the polypeptide of claim 8 comprising: contactingan analytically detectable ligand known to bind to the receptorpolypeptide and a compound with host cells expressing on the surfacethereof the polypeptide of claim 8, said polypeptide being associatedwith a second component capable of providing a detectable signal inresponse to the binding of a compound to said polypeptide underconditions to permit binding to the polypeptide; and determining whetherthe ligand binds to the polypeptide by detecting the absence of a signalgenerated from the interaction of the ligand with the polypeptide.
 19. Aprocess for diagnosing in a patient a disease or a susceptibility to adisease related to an under-expression of the polypeptide of claim 8comprising: determining a mutation in the nucleic acid sequence encodingsaid polypeptide, or the amount of the polypeptide in a sample derivedfrom a patient.
 20. A diagnostic process comprising: analyzing for thepresence of a soluble form of the polypeptide of claim 8 in a samplederived from a host.